Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized. Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks. Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D. Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

The bilayer disc is a flat, lipid aggregate structure in the nanometre regime. It is composed of a bilayer of amphiphilic molecules with micelle-forming amphiphilic molecules supporting the rim, which prevent disc fusion and self-closure. Stable discs have been found in lipid mixtures containing polyethylene glycol (PEG)-lipids as a rim-stabilizing component. One of the aims of the work described in this thesis was to increase the fundamental knowledge and understanding of the systems in which these discs are formed. Other micelle-forming surfactants apart from PEG-lipids were also explored to see if they could be used to stabilize the disc aggregate structure. Due to the similarities of these lipid discs with natural membranes it was hypothesized that they could be used as models for biological membranes. It was demonstrated that discs are formed in PEG-lipid/lipid systems when the lipid mixture contains components that reduce the spontaneous curvature and increase the monolayer bending rigidity. Discoidal structures are furthermore preferred when the lipids are in the gel phase, probably due to a combination of high bending rigidity and reduced PEG-lipid/lipid miscibility. The disc size could be varied by changing the PEG-lipid concentration. The size and size homogeneity of the discs could also be varied by changing the preparation path. Generally, the preferences of certain lipid systems to form discs remained when the PEG-lipid was replaced by more conventional surfactants. However, discs prepared in PEG-lipid/lipid systems are more useful as model membranes because of their relatively large size and good temperature, dilution and long-term stability. Data obtained with isothermal titration calorimetry and drug partition chromatography indicate that these bilayer discs may serve as an attractive and sometimes superior alternative to liposomes in studies of drug-membrane interactions.

<p>This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated.</p><p>The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol.</p><p>Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (T<i>C</i>). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through T<i>C</i> of the lipid mixture.</p><p>Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.</p>

This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated. The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol. Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (TC). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through TC of the lipid mixture. Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.

<p>When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions.</p><p>Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body.</p><p>Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks.</p><p>Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.</p>

When drug molecules are passively absorbed through the cell membrane in the small intestine, the first key step is partitioning of the drug into the membrane. Partition data can therefore be used to predict drug absorption. The partitioning of a solute can be analyzed by drug partition chromatography on immobilized model membranes, where the chromatographic retention of the solute reflects the partitioning. The aims of this thesis were to develop the model membranes used in drug partition chromatography and to study the effects of different membrane components and membrane structures on drug partitioning, in order to characterize drug–membrane interactions. Electrostatic effects were observed on the partitioning of charged drugs into liposomes containing charged detergent, lipid or phospholipid; bilayer disks; proteoliposomes and porcine intestinal brush border membrane vesicles (BBMVs), and on the retention of an oligonucleotide on positive liposomes. Biological membranes are naturally charged, which will affect drug partitioning in the human body. Proteoliposomes containing transmembrane proteins and cholesterol, BBMVs and bilayer disks were used as novel model membranes in drug partition chromatography. Partition data obtained on proteoliposomes and BBMVs demonstrated how cholesterol and transmembrane proteins interact with drug molecules. Such interactions will occur between drugs and natural cell membranes. In the use of immobilized BBMVs for drug partition chromatography, yet unsolved problems with the stability of the membrane were encountered. A comparison of partition data obtained on bilayer disks with data on multi- and unilamellar liposomes indicated that the structure of the membrane affect the partitioning. The most accurate partition values might be obtained on bilayer disks. Drug partition data obtained on immobilized model membranes include both hydrophobic and electrostatic interactions. Such partition data should preferably be used when deriving algorithms or computer programs for prediction of drug absorption.